Abstract
STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca2+ entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca2+ influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca2+ imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1−/−/Stim2−/− fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca2+ elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.
Highlights
Store-operated Ca2+ entry (SOCE) is an evolutionarily conserved signaling mechanism induced by the Ca2+ depletion of the endoplasmic reticulum (ER) that sustains long-lasting cytosolic Ca2+ signals required for transcription, cell proliferation and effector function (Hogan et al, 2010; Parekh, 2010)
A diffuse staining with few discrete fluorescence clusters was observed at rest, and new clusters appeared in the total internal reflection fluorescence (TIRF) plane following passive store depletion with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin in both STIM1- and STIM1L-expressing cells (Fig. 1A)
The amplitude of the thapsigargin-induced Ca2+ elevations was higher in YFP–STIM1L cells than in YFP–STIM1 cells, suggesting that the long isoform is more potent in mediating store-operated Ca2+ entry (SOCE)
Summary
Store-operated Ca2+ entry (SOCE) is an evolutionarily conserved signaling mechanism induced by the Ca2+ depletion of the endoplasmic reticulum (ER) that sustains long-lasting cytosolic Ca2+ signals required for transcription, cell proliferation and effector function (Hogan et al, 2010; Parekh, 2010). Upon ER Ca2+ depletion, Ca2+ dissociation from the STIM1 EFhand domain initiates the multimerization of STIM dimers into higher-order oligomers (Liou et al, 2005; Stathopulos et al, 2006) and induces conformational changes in the cytosolic domains that release the lysine-rich tail of STIM1 and expose the CAD (Covington et al, 2010; Korzeniowski et al, 2009; Luik et al, 2008; Muik et al, 2011; Zhang et al, 2005) This favors STIM1 translocation to the plasma membrane and the formation of STIM–Orai clusters at ER-plasma membrane junctions (Luik et al, 2006; Xu et al, 2006), where interactions between CAD and Orai N- and C-termini (Derler et al, 2013; Park et al, 2009; Zhou et al, 2010) promote pore opening and localized Ca2+ influx (Luik et al, 2006; Xu et al, 2006). These cortical ER cisternae, known as junctional ER, remain connected with the bulk ER and increase both in number and length upon ER Ca2+ depletion or STIM1 overexpression
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
